To understand the structural basis for the metal binding and activity of protein Mn and Fe sites, we have successfully initiated integrated mutant and structural studies of recombinant human Mn superoxide dismutase (SOD) and the di-Fe sites found in both the ribonucleotide reductase B2 subunit (RNRB2) and in myohemerythrin (MHr). The Fe and Mn SOD's, which have identical folds and active site geometries, are inactive if their metals are exchanged, suggesting that their highly similar active sites can differentiate between Fe and Mn for binding and for dismutation of the dioxygen superoxide radical. The RNRB2 and MHr di-Fe sites have structural similarities and both function in dioxygen binding, but only RNRB2 generates a stable Tyr radical for catalysis. We have cloned and overexpressed human MnSOD in E. coli, made and purified both wild-type and mutant enzymes for crystallographic and biochemical functional studies, and since January, have solved and analyzed the wild-type structure, solved one metal-site mutant and crystallized a second. We will also use the similar geometry for the Mn and Cu in the two different classes of SOD (Cu, Zn and Mn/Fe) to redesign MnSOD to bind Cu and again probe for structural and functional changes. to understand Fe binding and activity, including differences between the di-Fe sites in RNRB2 and MHr and between these di-Fe sites and the single Fe site in FeSOD, we have solved an initial RNRB2 structure in its active form and also cloned and expressed wild-type and mutant MHr proteins. These accomplishments establish the ability to complete the proposed comprehensive structure/function study of these metalloenzymes, using the techniques of molecular biology, biochemistry, X-ray crystallography, and computational analysis. We will use mutagenesis to redesign the metal binding sites in MnSOD, RNRB2 and MHr to probe metal ion binding specificity and activity for Mn, Fe, and Cu ions and test our redesign hypotheses by determining their atomic structures and biochemical activities and stabilities. For discovering mutants of MnSOD that maintain activity when Fe is substituted for Mn, and RNRB2 and MHr mutants remodeled to create SOD activity, we can use highly selective screening methods in E. coli strains lacking all wild-type Mn and FeSOD's. These comprehensive active site mutants will complement specific structure-based tests by identifying side chains with unexpected roles in metal binding and activity. In combination with the other 7 projects, the proposed research on these metalloenzymes is intended to probe structural determinants for their biological activity and for their recognition and discrimination of metal ions, elucidating the role of protein metal sites in the creation and dismutation of free radicals, and helping define the atomic basis for metal ion recognition and activity in proteins. These results will furthermore provide detailed structural data on the medically-important human MnSOD and RNR enzymes for the design of improved enzymes, inhibitors, and mimics, and for design of novel metalloproteins, such as metalloantibodies.